Carbamates of rapamycin

ABSTRACT

A compound of the structure ##STR1## wherein R and R 1  are each, independently, hydrogen, ##STR2## R 2  and R 3  are each, independently, hydrogen, alkyl, alkenyl, alkynyl, --CO 2  R 5 , --COR 5 , --CN, --NO 2 , --SO 2  R 5 , --SO 3  R 5 , --OR 5 , --SR 5 , or Ar; 
     R 4  is hydrogen, alkyl, alkenyl, alkynyl, --CF 3 , --NR 5  R 6 , --CO 2  R 5 , --COR 5 , CONR 5  R 6 , --NO 2 , halogen, --OR 5 , --SR 5 , --CN, --SO 2  R 5 , --SO 3  R 5 , --SO 2  NR 5  R 6  or Ar; 
     R 5  and R 6  are each, independently, hydrogen, alkyl, alkenyl, alkynyl, or Ar; ##STR3## are each, independently, a 5-7 membered saturated, unsaturated, or partially unsaturated heterocyclic radical, that is optionally fused to a phenyl ring or a cycloalkane or cycloalkene ring, wherein the heterocyclic ring may optionally contain O, S, or NR 8  in the heterocyclic ring, and may be optionally substituted by R 7  ; 
     R 7  is alkyl of 1-6 carbon atoms, alkenyl, alkynyl, --CF 3 , --NR 5  R 6 , --CO 2  R 5 , --COR 5 , CONR 5  R 6 , --NO 2 , halogen, --OR 5 , --SR 5 , --CN, --SO 2  R 5 , --SO 3  R 5 , --SO 2  NR 5  R 6 , or Ar; 
     R 8  is hydrogen, alkyl, alkenyl, alkynyl, --CF 3 , --NR 5  R 6 , --CO 2  R 5 , --COR 5 , CONR 5  R 6 , --OR 5 , --SR 5 , --CN, --SO 2  R 5 , --SO 3  R 5 , --SO 2  NR 5  R 6 , or Ar; 
     Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally substituted; with the proviso that R and R 1  are both not hydrogen, or a pharmaceutically acceptable salt thereof, which is useful as an immunosuppressive, antiinflammatory, antifungal, antiproliferative, and antitumor agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 08/259,701 filed Jun. 14,1994, now U.S. Pat. No. 5,434,260 which is a continuation of Ser. No.08/160,984, filed Dec. 1, 1993, now abandoned, which is a divisional ofSer. No. 08/054,655, filed Apr. 23, 1993 (now U.S. Pat. No. 5,302,584),which is a continuation-in-part of Ser. No. 07/960,597, filed Oct. 13,1992 now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to carbamates of rapamycin and a method for usingthem for inducing immunosuppression, and in the treatment oftransplantation rejection, graft vs. host disease, autoimmune diseases,diseases of inflammation, solid tumors, fungal infections, andhyperproliferative vascular disorders.

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus, which was found to have antifungal activity, particularlyagainst Candida albicans, both in vitro and in vivo [C. Vezina et al.,J. Antibiot. 28, 721 (1975); S. N. Sehgal et al., J. Antibiot. 28, 727(1975); H. A. Baker et al., J. Antibiot. 31,539 (1978); U.S. Pat. No.3,929,992; and U.S. Pat. No. 3,993,749].

Rapamycin alone (U.S. Pat. No. 4,885,171) or in combination withpicibanil (U.S. Pat. No. 4,401,653) has been shown to have antitumoractivity. R. Martel et al. [Can. J. Physiol. Pharmacol. 55, 48 (1977)]disclosed that rapamycin is effective in the experimental allergicencephalomyelitis model, a model for multiple sclerosis; in the adjuvantarthritis model, a model for rheumatoid arthritis; and effectivelyinhibited the formation of IgE-like antibodies.

The immunosuppressive effects of rapamycin have been disclosed in FASEB3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic molecules,also have been shown to be effective as immunosuppressive agents,therefore useful in preventing transplant rejection [FASEB 3, 3411(1989); FASEB 3, 5256 (1989); R. Y. Calne et al., Lancet 1183 (1978);and U.S. Pat. No. 5,100,899].

Rapamycin has also been shown to be useful in preventing or treatingsystemic lupus erythematosus [U.S. Pat. No. 5,078,999], pulmonaryinflammation [U.S. Pat. No. 5,080,899], insulin dependent diabetesmellitus [Fifth Int. Conf. Inflamm. Res. Assoc. 121 (Abstract), (1990)],and smooth muscle cell proliferation and intimal thickening followingvascular injury [Morris, R. J. Heart Lung Transplant 11 (pt. 2): 197(1992)].

Mono- and diacylated derivatives of rapamycin (esterified at the 28 and43 positions) have been shown to be useful as antifungal agents (U.S.Pat. No. 4,316,885) and used to make water soluble prodrugs of rapamycin(U.S. Pat. No. 4,650,803). Recently, the numbering convention forrapamycin has been changed; therefore according to Chemical Abstractsnomenclature, the esters described above would be at the 31- and 42-positions. U.S. Pat. No. 5,118,678 discloses carbamates of rapamycinthat are useful as immunosuppressive, anti-inflammatory, antifungal, andantitumor agents.

DESCRIPTION OF THE INVENTION

This invention provides derivatives of rapamycin which are useful asimmunosuppressive, antiinflammatory, antifungal, antiproliferative, andantitumor agents having the structure ##STR4## wherein R¹ and R² areeach, independently, hydrogen, --CONH--[(CR³ R⁴)_(m) (--A--(CR⁵R⁶)_(n))_(p) ]_(q) --B; ##STR5## R³, R⁴, R⁵, R⁶, and B are each,independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7carbon atoms, alkynyl of 2-7 carbon atoms, hydroxyalkyl of 1-6 carbonatoms, alkoxyalkyl of 2-12 carbon atoms, alkylthioalkyl of 2-12 carbonatoms, alkylaminoalkyl of 2-12 carbon atoms, dialkylaminoalkyl of 3-12carbon atoms, arylalkyl of 7-10 carbon atoms, cycloalkyl of 3-8 carbonatoms, --OR⁷, --SR⁷, halogen, --CN, --NO₂, --CF₃, --COR⁷, --CO₂ R⁷,--CONHR⁷, --SO₂ R⁷, --OSO₃ R⁷, --NR⁷ R⁸, --NHCOR⁷, --NHSO₂ R⁷, or Ar;

R⁷ and R⁸ are each, independently, hydrogen, alkyl of 1-6 carbon atoms,arylalkyl of 7-10 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of2-7 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12carbon atoms, alkylthioalkyl of 2-12 carbon atoms, alkylaminoalkyl of2-12 carbon atoms, dialkylaminoalkyl of 3-12 carbon atoms, cycloalkyl of3-8 carbon atoms, or Ar;

R⁹ and R¹⁰ are each, independently, alkyl of 1-6 carbon atoms, alkenylof 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, hydroxyalkyl of 1-6carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthioalkyl of 2-12carbon atoms, alkylaminoalkyl of 2-12 carbon atoms, dialkylaminoalkyl of3-12 carbon atoms, arylalkyl of 7-10 carbon atoms, cycloalkyl of 3-8carbon atoms, --CF₃, --COR⁷, --CO₂ R⁷, --CONHR⁷, --SO₂ R⁷, or Ar;

A is --CH₂ --, --NR⁷ --, --O--, --S--, --SO--, --SO₂ --, --PR⁷ --,--CO--, --NHCO--, --NHSO--, or --P(O)(R⁷)--;

Ar is phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, quinoxalyl,thienyl, thionaphthyl, furyl, benzofuryl, benzodioxyl, benzoxazolyl,benzoisoxazolyl, indolyl, thiazolyl, isoxazolyl, pyrimidinyl, pyrazinyl,imidazolyl, benzopyranyl, benz[b]thiophenolyl, benzimidazolyl,benzthiazolyl, benzodioxolyl, piperidinyl, morpholinyl, piperazinyl,tetrahydrofuranyl, or pyrrolidinyl; wherein the Ar group may beoptionally mono-, di-, or tri-substituted with a group selected fromalkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbonatoms, trifluoromethyl, amino, dialkylamino of 1-6 carbon atoms peralkyl group, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbonatoms, --SO₃ H, --PO₃ H, and --CO₂ H; ##STR6## is a nitrogen containingheterocycle that may be saturated, unsaturated, or partiallyunsaturated, and may be optionally mono-, di-, or tri- substituted witha group selected from alkyl of 1-6 carbon atoms, arylalkyl of 7-10carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro,carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, dialkylamino of1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 3-12 carbonatoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbonatoms, alkylthio of 1-6 carbon atoms, --SO₃ H, --PO₃ H, and --CO₂ H;

with the proviso that R¹ and R² are not both hydrogen;

m=0-6;

n=0-6;

p=0-1;

q=0-1;

or a pharmaceutically acceptable salt thereof.

The pharmaceutically acceptable salts are those derived from suchinorganic cations such as sodium, potassium, and the like; organic basessuch as: mono-, di-, and trialkyl amines of 1-6 carbon atoms, per alkylgroup and mono-, di-, and trihydroxyalkyl amines of 1-6 carbon atoms peralkyl group, and the like; and organic and inorganic acids as: acetic,lactic, citric, tartaric, succinic, maleic, malonic, gluconic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, and similarly known acceptable acids.

It is preferred that the aryl portion of the arylalkyl substituent is aphenyl, piperazinyl, piperidinyl, or pyridyl group that is optionallymono-, di-, or tri-substituted with a group selected from alkyl of 1-6carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbonatoms, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms,trifluoromethyl, amino, dialkylamino of 1-6 carbon atoms per alkylgroup, alkylthio of 1-6 carbon atoms, --SO₃ H, --PO₃ H, and --CO₂ H. Theterm alkyl includes both straight chain and branched alkyl groups.

It is preferred that ##STR7## is a pyridyl, pyrazinyl, piperidinyl,morpholinyl, piperazinyl, pyrrolidinyl, thiazolyl, pyrimidinyl,isoxazolyl, pyrrolidinyl, or imidazolyl group that may be optionallysubstituted as described above.

This invention also discloses preferred amidino carbamates having thestructure ##STR8## wherein R and R¹ are each, independently, hydrogen,##STR9## R² and R³ are each, independently, hydrogen, alkyl of 1-6carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,--CO₂ R⁵, --COR⁵, --CN, --NO₂, --SO₂ R⁵, --SO₃ R⁵, --OR⁵, --SR⁵, or Ar;

R⁴ is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,alkynyl of 2-7 carbon atoms, --CF_(3l) , --NR⁵ R⁶, --CO₂ R⁵, --COR⁵,CONR⁵ R⁶, --NO₂, halogen, --OR⁵, --SR⁵, --CN, --SO₂ R⁵, --SO₃ R⁵, --SO₂NR⁵ R⁶, or Ar;

R⁵ and R⁶ are each, independently, hydrogen, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar;##STR10## are each, independently, a 5-7 membered saturated,unsaturated, or partially unsaturated heterocyclic radical, that isoptionally fused to a phenyl ring or a cycloalkane or cycloalkene ringof 5-7 carbon atoms, wherein the heterocyclic ring may optionallycontain O, S, or NR⁸ in the heterocyclic ting, and may be optionallysubstituted by R⁷ ;

R⁷ is alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of2-7 carbon atoms, --CF₃, --NR⁵ R⁶, --CO₂ R⁵, --COR⁵, CONR⁵ R⁶, --NO₂,halogen, --OR⁵, --SR⁵, --CN, --SO₂ R⁵, --SO₃ R⁵, --SO₂ NR⁵ R⁶, or Ar;

R⁸ is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,alkynyl of 2-7 carbon atoms, --CF₃, --NR⁵ R⁶, --CO₂ R⁵, --COR⁵, CONR⁵R⁶, --OR⁵, --SR⁵, --CN, --SO₂ R⁵, --SO₃ R⁵, --SO₂ NR⁵ R⁶, or Ar;

Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may beoptionally mono-, di-, or tri-substituted with a group selected fromalkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbonatoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbon atoms,trifluoromethyl, trifluoromethoxy, amino, dialkylamino of 1-6 carbonatoms per alkyl group, dialkylaminoalkyl of 3-12 carbon atoms,hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon atoms,alkylthio of 1-6 carbon atoms, --SO₃ H, and --CO₂ H;

with the proviso that R and R¹ are both not hydrogen, or apharmaceutically acceptable salt thereof.

For the compounds having structure II (immediately above), the termsalkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, and alkynyl of2-7 carbon atoms, include both straight chain as well as branched carbonchains. When any of the genetic terms (i.e., R⁵) are contained more thanonce in a given compound, each may be the same or different. Thepharmaceutically acceptable salts of the compounds having structure IIare the same as was defined following the compounds of structure I.

For the compounds having structure II, hetaryl is defined as anunsaturated or partially saturated heterocyclic radical of 5-12 atomshaving 1 ring or 2 fused rings. Preferred heterocyclic radicals includeunsaturated heterocyclic radicals such as furanyl, thiophenyl, pyrrolyl,pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2dithiolyl,1,3-dithiolyl, 1,2,3-oxathiolyl, isoxazolyl, oxazolyl, thiazolyl,isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,3,4-oxatriazolyl, 1,2,3,5-oxatriazolyl, 1,2,3-dioxazolyl,1,2,4-dioxazolyl, 1,3,2-dioxazolyl, 1,3,4-dioxazolyl,1,2,5-oxathiazolyl, 1,3-oxathiolyl, 1,2-pyranyl, 1,4-pyranyl, pyridinyl,pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, 1,2,4-triazinyl,1,2,3-triazinyl, 1,2,4-oxazinyl, 1,3,2-oxazinyl, 1,2,6-oxazinyl,1,4-oxazinyl, isoxazinyl, 1,2,5-oxathiazinyl, 1,4-oxazinyl,o-isoxazinyl, p-isoxazinyl, 1,2,5-oxathiazinyl, 1,2,6-oxathiazinyl,1,3,5,2-oxadiazinyl, azepinyl, oxepinyl, thiepinyl, 1,2,4-diazepinyl,benzofuranyl, isobenzofuranyl, thionaphthenyl, indolyl, indolenyl,2-isobenzazolyl, 1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, benzpyrazolyl,benzisoxazolyl, benzoxazolyl, anthranilyl, 1,2-benzopyranyl, quinolinyl,isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl,pyrido[3,4-b]pyridinyl, pyrido[4,3-b]pyridinyl, pyrido[2,3-b]pyridinyl,1,3,2-benzozazinyl, 1,4,2-benzoxazinyl, 2,3,1-benzoxazinyl,3,1,4-benzoxazinyl, 1,2-benzisoxazinyl, 1,4-benzisoxazinyl, carbazolyl,purinyl, and partially saturated heterocyclic radicals selected from thelist above. All of the preferred heterocyclic radicals contain at leastone double bond. When the heterocyclic radical is partially saturated,one or more of the olefins in the unsaturated ring system is saturated;the partially saturated heterocyclic radical still contains at least onedouble bond. It is more preferred that hetaryl is pyridinyl.

For the amidino carbamates having structure II, it is preferred that the##STR11## moieties are pyrrolyl, 3,4-dihydropyrrolyl, pyrrolidinyl,pyrazolyl, imidazolyl, 4,5-dihydroimidazolyl, 2-pyrazolinyl,1,2,4-triazolyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl,pyridinyl, 1,2,3,6-tetrahydropyridinyl, piperidinyl, pyridazinyl,pyrimidinyl, 1,4,5,6-tetrahydropyrimidinyl, pyrazinyl, piperazinyl,1,3,5-triazinyl, hexahydro-1,3,5-triazinyl, 1,2,4-triazinyl,1,3,2-oxazinyl, 1,4-oxazinyl, morpholinyl, azepinyl, indolyl, indolinyl,indolenyl, benzoxazolyl, quinolinyl, 1,2,3,4-tetrahydroquinolinyl,5,6,7,8-tetrahydroquinolinyl, isoquinolinyl, 3,4-dihydroisoquinolinyl,1,2,3,4-tetrahydroisoquinolinyl, and, 5,6,7,8-tetrahydroisoquinolinylradicals that are optionally substituted by R⁸. The fourth ringstructure, above, is the more preferred of the four ring systems, andwithin this ring system it is more preferred that the radical is4,5-dihydroimidazolyl or 1,4,5,6-tetrahydropyrimidinyl. It is alsopreferred that the R⁷ substituent, when present, is phenyl.

The compounds of this invention carbamylated at the 42-position or atboth the 31- and 42-positions can be prepared by convening the 42-and/or 31-alcohols of rapamycin to a carbonate (see Example 1 ) followedby reaction with an appropriately substituted amine to provide thedesired carbamate. The following scheme illustrates the preparation ofthe compound of Example 4. ##STR12##

EXAMPLE 4

Alternatively, the compounds of this invention carbamylated at the42-position or at both the 3 1- and 42-positions can be prepared byreacting rapamycin with an appropriately substituted isocyanate underneutral conditions or in the presence of a base, such as pyridine.Preparation of carbamates of rapamycin using this method was disclosedin U.S. Pat. No. 5,118,678, which is hereby incorporated by reference.The 31-carbamylated compounds of this invention can be prepared byprotecting the 42-alcohol of rapamycin with a protecting group, such aswith a tert-butyl dimethylsilyl group, followed by carbamylation of the31-position by the procedures described above. Removal of the protectinggroup provides the 31-carbamylated compounds. In the case of thetert-butyl dimethylsilyl protecting group, deprotection can beaccomplished under mildly acidic conditions.

Having the 31-position carbamylated and the 42-position deprotected, the42-position can be carbamylated using a different amine (via thecarbonate) or isocyanate than was reacted with the 31-alcohol, to givecompounds having different carbamates at the 31- and 42- positions.Alternatively, the 42-carbamylated compounds, prepared as describedabove, can be reacted with a different amine (via the carbonate) orisocyanate to provide compounds having different carbamates at the 31-and 42-positions.

The amines and isocyanates used to prepare the compounds of theinvention are commercially available or can be prepared by methods thatare disclosed in the literature.

This invention also covers analogous carbamates of other rapamycins suchas, but not limited to, 29-demethoxyrapamycin, [U.S. Pat. No. 4,375,464,32-demethoxyrapamycin under C.A. nomenclature]; rapamycin derivatives inwhich the double bonds in the 1-, 3-, and/or 5-positions have beenreduced [U.S. Pat. No. 5,023,262]; 42-oxorapamycin [U.S. Pat. No.5,023,262]; 29-desmethylrapamycin [U.S. Pat. No. 5,093,339,32-desmethylrapamycin under C.A. nomenclature];7,29-bisdesmethylrapamycin [U.S. Pat. No. 5,093,338,7,32-desmethylrapamycin under C.A. nomenclature]; and 15-hydroxy- and15,27-bishydroxy-rapamycin [U.S. Pat. No. 5,102,876]. The disclosures inthe above cited U.S. Patents are hereby incorporated by reference.

Immunosuppressive activity for representative compounds of thisinvention was evaluated in an in vitro. standard pharmacological testprocedure to measure lymphocyte proliferation (LAF) and in an in vivostandard pharmacological test procedure which evaluated the survivaltime of a pinch skin graft.

The comitogen-induced thymocyte proliferation procedure (LAF) was usedas an in vitro measure of the immunosuppressive effects ofrepresentative compounds. Briefly, cells from the thymus of normalBALB/c mice are cultured for 72 hours with PHA and IL-1 and pulsed withtritiated thymidine during the last six hours. Cells are cultured withand without various concentrations of rapamycin, cyclosporin A, or testcompound. Cells are harvested and incorporated radioactivity isdetermined. Inhibition of lymphoproliferation is assessed as percentchange in counts per minute from non-drug treated controls. For eachcompound evaluated, rapamycin was also evaluated for the purpose ofcomparison. An IC₅₀ was obtained for each test compound as well as forrapamycin. When evaluated as a comparator for the representativecompounds of this invention, rapamycin had an IC₅₀ ranging from 0.3-0.9nM. The results obtained are provided as an IC₅₀ and as the percentinhibition of T-cell proliferation at 0.1 μM. The results obtained forthe representative compounds of this invention were also expressed as aratio compared with rapamycin. A positive ratio indicatesimmunosuppressive activity. A ratio of greater than 1 indicates that thetest compound inhibited thymocyte proliferation to a greater extent thanrapamycin. Calculation of the ratio is shown below. ##EQU1##

Representative compounds of this invention were also evaluated in an invivo test procedure designed to determine the survival time of pinchskin graft from male BAB/c donors transplanted to male C₃ H(H-2K)recipients. The method is adapted from Billingham R. E. and Medawar P.B., J. Exp. Biol. 28:385-402, (1951). Briefly, a pinch skin graft fromthe donor was grafted on the dorsum of the recipient as a allograft, andan isograft was used as control in the same region. The recipients weretreated with either varying concentrations of test compoundsintraperitoneally or orally. Rapamycin was used as a test control.Untreated recipients serve as rejection control. The graft was monitoreddaily and observations were recorded until the graft became dry andformed a blackened scab. This was considered as the rejection day. Themean graft survival time (number of days ±S.D.) of the drug treatmentgroup was compared with the control group. The following table shows theresults that were obtained. Results are expressed as the mean survivaltime in days. Untreated (control) pinch skin grafts are usually rejectedwithin 6-7 days. The results shown in Table 1 are based on a dose of 4mg/kg of test compound. A survival time of 12.0±1.7 days was obtainedfor rapamycin at 4 m g/kg.

The following table summarizes the results of representative compoundsof this invention in these two standard test procedures.

                  TABLE 1                                                         ______________________________________                                        EVALUATION OF IMMUNOSUPPRESSIVE ACTIVITY*                                               LAF           Skin Graft                                            Compound  IC.sub.50 (nM)                                                                              (ratio) (days ± SD)                                ______________________________________                                        Example 2 0.54          0.63    10.8 ± 1.0                                 Example 3 1.70          0.20     9.8 ± 1.5                                 Example 4 1.21          0.78                                                  ______________________________________                                         *Calculation of the ratio was described supra.                           

The results of these standard pharmacological test proceduresdemonstrate immunosuppressive activity both in vitro and in vivo for thecompounds of this invention. Positive ratios in the LAF test proceduresindicates suppression of T-cell proliferation, thereby demonstrating theimmunosuppressive activity of the compounds of this invention. Astransplanted pinch skin grafts are typically rejected within 6-7 dayswithout the use of an immunosuppressive agent, the increased survivaltime of the skin graft when treated with the compounds of this inventionfurther demonstrates their utility as immunosuppressive agents.Additionally, the results obtained in the skin graft test procedurefurther demonstrates the ability of the compounds of this invention totreat or inhibit transplantation rejection.

Based on the results of these standard pharmacological test procedures,the compounds are useful in the treatment or inhibition oftransplantation rejection such as kidney, heart, liver, lung, bonemarrow, pancreas (islet cells), cornea, small bowel, and skinallografts, and heart valve xenografts; in the treatment or inhibitionof graft vs. host disease; in the treatment or inhibition of autoimmunediseases such as lupus, rheumatoid arthritis, diabetes mellitus,myasthenia gravis, and multiple sclerosis; and diseases of inflammationsuch as psoriasis, dermatitis, eczema, seborrhea, inflammatory boweldisease, pulmonary inflammation (including asthma, chronic obstructivepulmonary disease, emphysema, acute respiratory distress syndrome,bronchitis, and the like), and eye uveitis.

Because of the activity profile obtained, the compounds of thisinvention also are considered to have antitumor, antifungal activities,and antiproliferative activities. The compounds of this inventiontherefore also useful in treating solid tumors, adult T-cellleukemia/lymphoma, fungal infections, and hyperproliferative vasculardiseases such as restenosis and atherosclerosis. When used forrestenosis, it is preferred that the compounds of this invention areused to treat restenosis that occurs following an angioplasty procedure.When used for this purpose, the compounds of this invention can beadministered prior to the procedure, during the procedure, subsequent tothe procedure, or any combination of the above.

When administered for the treatment or inhibition of the above diseasestates, the compounds of this invention can be administered to a mammalorally, parenteraly, intranasally, intrabronchially, transdermaly,topically, intravaginaly, or rectally.

It is contemplated that when the compounds of this invention are used asan immunosuppressive, antirejection, or antiinflammatory agent, they canbe administered in conjunction with one or more other immunoregulatoryagents. Such other immunoregulatory agents include, but are not limitedto azathioprine, corticosteroids, such as prednisone andmethylprednisolone, cyclophosphamide, rapamycin, cyclosporin A, FK-506,OKT-3, and ATG. By combining the compounds of this invention with suchother drugs or agents for inducing immunosuppression or treatinginflammatory conditions, lesser amounts of each of the agents arerequired to achieve the desired effect. The basis for such combinationtherapy was established by Stepkowski whose results showed that the useof a combination of rapamycin and cyclosporin A at subtherapeutic dosessignificantly prolonged heart allograft survival time. [TransplantationProc. 23:507 (199 1 )].

The compounds of this invention can be formulated neat or with apharmaceutical carrier to a mammal in need thereof. The pharmaceuticalcarrier may be solid or liquid. When formulated orally, it has beenfound that 0.01% Tween 80 in PHOSAL PG-50 (phospholipid concentrate with1,2-propylene glycol, A. Nattermann & Cie. GmbH) provides an acceptableoral formulation.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is aFreely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

The compounds of this invention may be administered rectally in the formof a conventional suppository. For administration by intranasal orintrabronchial inhalation or insufflation, the compounds of thisinvention maybe formulated into an aqueous or partially aqueoussolution, which can then be utilized in the form of an aerosol. Thecompounds of this invention may also be administered transdermallythrough the use of a transdermal patch containing the active compoundand a carrier that is inert to the active compound, is non toxic to theskin, and allows delivery of the agent for systemic absorption into theblood stream via the skin. The carrier may take any number of forms suchas creams and ointments, pastes, gels, and occlusive devices. The creamsand ointments may be viscous liquid or semisolid emulsions of either theoil-in-water or water-in-oil type. Pastes comprised of absorptivepowders dispersed in petroleum or hydrophilic petroleum containing theactive ingredient may also be suitable. A variety of occlusive devicesmay be used to release the active ingredient into the blood stream suchas a semipermiable membrane covering a reservoir containing the activeingredient with or without a carrier, or a matrix containing the activeingredient. Other occlusive devices are known in the literature.

In addition, the compounds of this invention may be employed as asolution, cream, or lotion by formulation with pharmaceuticallyacceptable vehicles containing 0.1-5 percent, preferably 2%, of activecompound which may be administered to a fungally affected area.

The dosage requirements vary with the particular compositions employed,the route of administration, the severity of the symptoms presented andthe particular subject being treated. Based on the results obtained inthe standard pharmacological test procedures, projected daily dosages ofactive compound would be 0.1 μg/kg - 100 mg/kg, preferably between0.001-25 mg/kg, and more preferably between 0.01-5 mg/kg. Treatment willgenerally be initiated with small dosages less than the optimum dose ofthe compound. Thereafter the dosage is increased until the optimumeffect under the circumstances is reached; precise dosages for oral,parenteral, nasal, or intrabronchial administration will be determinedby the administering physician based on experience with the individualsubject treated. Preferably, the pharmaceutical composition is in unitdosage form, e.g. as tablets or capsules. In such form, the compositionis sub-divided in unit dose containing appropriate quantities of theactive ingredient; the unit dosage forms can be packaged compositions,for example, packeted powders, vials, ampoules, prefilled syringes orsachets containing liquids. The unit dosage form can be, for example, acapsule or tablet itself, or it can be the appropriate number of anysuch compositions in package form.

The following examples illustrate the preparation of representativecompounds of this invention.

EXAMPLE 1 42-O-(4-Nitro-phenoxycarbonyl)rapamycin

To a solution of 5.15 g (5.633 mmol) of rapamycin in 40 ml of methylenechloride cooled to -78° C. with dry ice/acetone bath, was added 0.7 mldry pyridine and 1.70 g (8.450 mmol) of p-nitrophenylchloroformatedissolved in 10 ml methylene chloride. The reaction mixture was allowedto warm to ambient and stirred overnight under nitrogen. The reactionmixture was concentrated in vacuo and partitioned between ether andwater. The organic phase was washed with 0.1 N HCl (3×), then with asaturated sodium chloride solution (2×), dried over magnesium sulfate,filtered and concentrated under vacuum to give a pale yellow solid.Purification by flash column chromatography (elution with 40% then 50%ethyl acetate/hexanes) gave 5.41 g (88 %) of the title compound as apale yellow solid. ¹ H NMR (DMSO) d 8.3 and 7.5 (aromatic-H, 4H), 4.5(42C-H, 1H). MS (-) FAB m/z: 1078 (M⁻), 590 (Southern Fragment).

EXAMPLE 2 Rapamycin 42-ester with5-phenyl-5,6-dihydro-4H-pyrimidine-1-carboxylic acid

To 0.5716 g (2.779 mmol) of 5-phenyl-1,4,5,6-tetrahydro-pyrimidinehydrochloride hemihydrate was added one equivalent of 0.1M sodiumhydroxide/methanol after which the solvent was removed in vacuo. To thesolution of the free base in 15 ml of DMF was added 3.00 g (2.779 mmol)of 42-O-(4-Nitro-phenoxycarbonyl)rapamycin. The reaction mixture wasallowed to stir under nitrogen for 5 hours at ambient temperature, thenwas diluted with ethyl acetate and washed with portions of H₂ O andbrine. The organic phase was dried over magnesium sulfate, filtered andconcentrated under vacuum to yield crude product. Purification by flashcolumn chromatography (elution with 100% ethyl acetate(2×)) gave 1.8157g (59%) of the title compound as a cream colored solid. ¹ H NMR (DMSO)δ8.0 (s, CH═N, 1H), 7.3 (m, aromatic-H, 5H), 4.54 (m, 42C-H, 1H), 3.5(m, CH₂ N, CH₂ N═C, 4H). MS (-) FAB m/z: 1099 (M⁻), 590 (SouthernFragment), 507 (Northern Fragment).

EXAMPLE 3 Rapamycin 42-ester with5,6-dihydro-4-H-pyrimidine-1-carboxylic acid

To a solution of 3.00 g (2.229 mmol) of42-O-(4-Nitro-phenoxycarbonyl)rapamycin in 15 ml of DMF was added 0.25ml (3.043 mmol) of 1,4,5,6-tetrahydropyrimidine. The reaction mixturewas allowed to stir under nitrogen for 8 hours at ambient temperature,then was diluted with ethyl acetate and washed with portions of H₂ O andbrine. The organic phase was dried over magnesium sulfate, filtered andconcentrated under vacuum to yield crude product. Purification by flashcolumn chromatography (eluting 7.5% then 10% isopropanol/methylenechloride) gave 0.403 g (14%) of the title compound as a pale yellowsolid. ¹ H NMR (DMSO) δ7.8 (s, CH═N, 1H), 4.5 (m, 42C-H, 1H), 3.5 (m,CH₂ -N, 2H), 3.3 (m, CH₂ --N═, 2H), 1.6 (m, CH₂, 2H). MS (-) FAB m/z:1023 (M⁻), 590 (Southern Fragment), 43 1 (Northern Fragment).

EXAMPLE 4 Rapamycin 42-ester with2-phenyl-4,5-dihydroimidazole-1-carboxylic acid

To a solution of 5.0058 g (4.638 mmol) of42-O-(4-Nitro-phenoxycarbonyl)rapamycin in 25 ml of DMF was added 0.6782g (4.639 mmol) of 2-phenyl-2-imidazoline. The reaction mixture wasallowed to stir under nitrogen for 9 hours at ambient temperature, thenwas diluted with ethyl acetate and washed with portions of H₂ O andbrine. The organic phase was dried over magnesium sulfate, filtered andconcentrated under vacuum to yield crude product. Purification by flashcolumn chromatography (eluting 100% ethyl acetate/hexanes (2×) followedwith 7.5% isopropanol/methylene chloride) gave 0.6064 g (16 %) of thetitle compound as a white solid. ¹ H NMR (DMSO) δ7.48-7.35 (m,aromatic-H, 5H), 3.88 (m, CH₂ CH₂ N═C, 4H), 4.35 (m, 42C-H, 1H). MS (-)FAB m/z: 1085 (M⁻), 590 (Southern Fragment), 493 (Northern Fragment).Results obtained in standard pharmacological test procedures:

What is claimed is:
 1. A compound of the structure ##STR13## wherein Rand R¹ are each, independently, hydrogen, ##STR14## R² and R³ are each,independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7carbon atoms, alkynyl of 2-7 carbon atoms, --CO₂ R⁵, --COR⁵, --CN,--NO₂, --SO₂ R⁵, --SO₃ R⁵, --OR⁵, --SR⁵, or Ar;R⁴ is hydrogen, alkyl of1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbonatoms, --CF₃, --NR⁵ R⁶, --CO₂ R⁵, --COR⁵, CONR⁵ R⁶, --NO₂, halogen,--OR⁵, --SR⁵, --CN, --SO₂ R⁵, --SO₃ R⁵, --SO₂ NR⁵ R⁶, or Ar; R⁵ and R⁶are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar; ##STR15## areeach, independently, a heterocyclic radical selected from the groupconsisting of pyrrolyl, 3,4-dihydropyrrolyl, pyrrolidinyl, pyrazolyl,imidazolyl, 4,5-dihydroimidazolyl, 2-pyrazolinyl, 1,2,4-triazolyl,isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, pyridinyl,1,2,3,6-tetrahydropyridinyl, piperidinyl, pyridazinyl, pyrimidinyl,1,4,5,6-tetrahydropyrimidinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl,hexahydro-1,3,5-triazinyl, 1,2,4-triazinyl, 1,3,2-oxazinyl,1,4-oxazinyl, morpholinyl, azepinyl, indolyl, indolinyl, indolenyl,benzoxazolyl, quinolinyl, 1,2,3,4-tetrahydroquinolinyl,5,6,7,8-tetrahydroquinolinyl, isoquinolinyl, 3,4-dihydroisoquinolinyl,1,2,3,4-tetrahydroisoquinolinyl, and 5,6,7,8-tetrahydroisoquinolinyl,wherein the heterocyclic radical may be optionally substituted by R⁷ ;R⁷ is alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of2-7 carbon atoms, --CF₃, --NR⁵ R⁶, --CO₂ R⁵, --COR⁵, CONR⁵ R⁶, --NO₂,halogen, --OR⁵, --SR⁵, --CN, --SO₂ R⁵, --SO₃ R⁵, --SO₂ NR⁵ R⁶, or Ar; Aris phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionallymono-, di-, or tri-substituted with a group selected from alkyl of 1-6carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo,hydroxy, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl,trifluoromethoxy, amino, dialkylamino of 1-6 carbon atoms per alkylgroup, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbonatoms, --SO₃ H, and --CO₂ H; hetaryl is a heterocyclic radical selectedfrom the group consisting of furanyl, thiophenyl, pyrrolyl, pyrazolyl,imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2-dithiolyl,1,3-dithiolyl, 1,2,3-oxathiolyl, isoxazolyl, oxazolyl, thiazolyl,isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,3,4-oxatriazolyl, 1,2,3,5-oxatriazolyl, 1,2,3-dioxazolyl,1,2,4-dioxazolyl, 1,3,2-dioxazolyl, 1,3,4-dioxazolyl,1,2,5-oxathiazolyl, 1,3-oxathiolyl, 1,2-pyranyl, 1,4-pyranyl, pyridinyl,pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, 1,4-triazinyl,1,2,3-triazinyl, 1,2,4-oxazinyl, 1,3,2-oxazinyl, 1,2,6-oxazinyl,1,4-oxazinyl, isoxazinyl, 1,2,5-oxathiazinyl, 1,4-oxazinyl,o-isoxazinyl, p-isoxazinyl, 1,2,5-oxathiazinyl, 1,2,6-oxathiazinyl,1,3,5,2-oxadiazinyl, azepinyl, oxepinyl, thiepinyl, 1,2,4-diazepinyl,benzofuranyl, isobenzofuranyl, thionaphthenyl, indolyl, indolenyl,2-isobenzazolyl, 1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, benzpyrazolyl,benzisoxazolyl, benzoxazolyl, anthranilyl, 1,2-benzopyranyl, quinolinyl,isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl,pyrido[3,4-b]pyridinyl, pyrido[4,3-b]pyridinyl, pyrido[2,3-b]pyridinyl,1,3,2-benzozazinyl, 1,4,2-benzoxazinyl, 2,3,1-benzoxazinyl,3,1,4-benzoxazinyl, 1,2-benzisoxazinyl, 1,4-benzisoxazinyl, carbazolyl,purinyl, and a partially saturated heterocyclic radical selected fromthe foregoing; wherein the partially saturated heterocyclic radicalcontains at least one double bond: with the proviso that R and R¹ areboth not hydrogen, or a pharmaceutically acceptable salt thereof.
 2. Thecompound according to claim 1, wherein R¹ is hydrogen or apharmaceutically acceptable salt thereof.
 3. The compound according toclaim 2 wherein R is ##STR16## or a pharmaceutically acceptable saltthereof.
 4. The compound according to claim 3 wherein the ##STR17##moiety is a 4,5-dihydroimidazolyl or 1,4,5,6-tetrahydropyrimidinylradical that is optionally substituted by R⁷ or a pharmaceuticallyacceptable salt thereof.
 5. The compound according to claim 4, whereinR⁷ is phenyl or a pharmaceutically acceptable salt thereof.
 6. Thecompound according to claim 1 which is rapamycin 42-ester with5-phenyl-5,6-dihydro-4H-pyrimidine-1-carboxylic acid or apharmaceutically acceptable salt thereof.
 7. The compound according toclaim 1 which is rapamycin 42-ester with5,6-dihydro-4-H-pyrimidine-1-carboxylic acid or a pharmaceuticallyacceptable salt thereof.
 8. The compound according to claim 1 which israpamycin 42-ester with 2-phenyl-4,5-dihydroimidazole-1-carboxylic acidor a pharmaceutically acceptable salt thereof.